Triple-negative breast cancer (TNBC) is a subset of breast cancers with poor prognosis and high mortality due to the lack of effective therapeutic regimens. This Phase I SBIR application is to develop E1-Rap, a novel immunoRNases, for treating TNBC. E1-Rap is a DOCK-AND-LOCKTM (DNLTM) complex, comprising four copies of ranpirnase (Rap) site-specifically tethered to the CH3-terminus of hRS7, a humanized monoclonal antibody targeting TROP-2, a transmembrane protein over-expressed in diverse epithelial cancers. We have shown that TROP-2 is present on the surface of TNBC cells, and targeted delivery of E1-Rap significantly enhances the binding, internalization, and cytotoxicity of Rap in TROP-2-positive cell lines. More importantly, E1-Rap has greatly improved the potency over the previously made fusion protein (Rap-hRS7), but maintained minimal toxicity to TROP-2-negative cell lines and human PBMC. In this Phase I application, we will scale up the production of E1-Rap and evaluate the in vivo properties of E1-Rap, including PK, stability, safety, bioavailability, and the therapeutic activity of E1-Rap in human TNBC xenograft models. Successful accomplishments of these Phase I goals will lead to a Phase II application aimed to complete the preclinical development of E1-Rap for clinical trials.